Encochleation methods, cochleates and methods of use

Abstract

Disclosed are novel methods for making cochleates and cochleate compositions that include introducing a cargo moiety to a liposome in the presence of a solvent. Also disclosed are cochleates and cochleate compositions that include an aggregation inhibitor, and optionally, a cargo moiety. Additionally, anhydrous cochleates that include a protonized cargo moiety, a divalent metal cation and a negatively charge lipid are disclosed. Methods of using the cochleate compositions of the invention, including methods of administration, are also disclosed.

We claim: 
     
       1. A method for forming a cochleate comprising a cargo moiety, wherein the method comprises:
 (a) solubilizing the cargo moiety in a water miscible solvent; 
 (b) suspending a mixture of lipids in an aqueous solution to form a liposomal suspension comprising liposomes, wherein a majority of the mixture of lipids comprises a negatively charged phospholipid; 
 (c) combining the cargo moiety and the liposomes; 
 (d) precipitating the liposomes with a multivalent cation to form the cochleate comprising the cargo moiety; and 
 (e) removing the water-miscible solvent from the cochleate formed in step (d), 
 wherein the cargo moiety is protonized in any one or more of steps (a), (b), (c), and (d), by addition of an acid thereby lowering the pH of the solution below a maximum pK a  of the cargo moiety. 
 
     
     
       2. The method of  claim 1 , wherein the cargo moiety is added by dropwise addition, continuous flow addition, or in a bolus. 
     
     
       3. The method of  claim 1 , wherein the cargo moiety is introduced into the water miscible solvent in the form of a powder or a liquid. 
     
     
       4. The method of  claim 1 , where an antioxidant is introduced into the liposomal suspension. 
     
     
       5. The method of  claim 1 , wherein the liposomal suspension comprises a plurality of unilamellar and multilamellar liposomes. 
     
     
       6. The method of  claim 1 , further comprising the step of filtering or mechanically extruding through a small aperture the liposomal suspension such that a majority of the liposomes are unilamellar. 
     
     
       7. The method of  claim 1 , wherein the water miscible solvent is at least one of dimethylsulfoxide (DMSO), a methylpyrrolidone, N-methylpyrrolidone (NMP), acetonitrile, alcohol, ethanol, dimethylformamide (DMF), ethanol (EtOH), tetrahydrofuran (THF), and combinations thereof. 
     
     
       8. The method of  claim 1 , wherein the water-miscible solvent is removed from the cochleate by dialysis or washing. 
     
     
       9. The method of  claim 1 , wherein the ratio of the lipid to the cargo moiety is between about 0.5:1 and about 20:1. 
     
     
       10. The method of  claim 1 , wherein the ratio of the lipid to the cargo moiety is between about 20:1 and about 20,000:1. 
     
     
       11. The method of  claim 1 , wherein the cargo moiety is hydrophobic or hydrophilic or hydrosoluble. 
     
     
       12. The method of  claim 1 , wherein the cargo moiety is amphipathic. 
     
     
       13. The method of  claim 1 , wherein the cargo moiety is an antifungal agent. 
     
     
       14. The method of  claim 1 , wherein the cargo moiety is at least one member selected from the group consisting of a vitamin, a mineral, a nutrient, a micronutrient, an amino acid, a toxin, a microbicide, a microbistat, a co-factor, an enzyme, a polypeptide, a polypeptide aggregate, a polynucleotide, a lipid, a carbohydrate, a nucleotide, a starch, a pigment, a fatty acid, a saturated fatty acid, a monounsaturated fatty acid, a polyunsaturated fatty acid, a flavoring, an essential oil or extract, a hormone, a cytokine, a virus, an organelle, a steroid or other multi-ring structure, a saccharide, a metal, a metabolic poison, an antigen, an imaging agent, a porphyrin, a tetrapyrrolic pigment, and a drug. 
     
     
       15. The method of  claim 14 , wherein the drug is at least one of a protein, a small peptide, a bioactive polynucleotide, an antibiotic, an antiviral, an anesthetic, antipsychotic, an anti-infectious, an antifungal, an anticancer, an immunosuppressant, an immunostimulant, a steroidal anti-inflammatory, a non-steroidal anti-inflammatory, an antioxidant, an antidepressant which can be synthetically or naturally derived, a substance which supports or enhances mental function or inhibits mental deterioration, an anticonvulsant, an HIV protease inhibitor, a non-nucleophilic reverse transcriptase inhibitor, a cytokine, a tranquilizer, a mucolytic agent, a dilator, a vasoconstrictor, a decongestant, a leukotriene inhibitor, an anti-cholinergic, an anti-histamine, a cholesterol lipid metabolism modulating agent, or a vasodilatory agent. 
     
     
       16. The method of  claim 14 , wherein the drug is at least one of vancomycin, teicoplanin, bleomycin, peptidolglycan, ristocetin, sialoglycoproteins, orienticin, avaporcin, helevecardin, galacardin, actinoidin, gentamycin, netilmicin, tobramycin, amikacin, kanamycin A, kanamycin B, neomycin, paromomycin, neamine, streptomycin, dihydrostreptomycin, apramycin, ribostamycin, and spectinomycin. 
     
     
       17. The method of  claim 14 , wherein the polynucleotide is at least one member selected from the group consisting of a deoxyribonucletic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, small interfering RNA (siRNA), a ribozyme, an antisense molecule, a morpholino and a plasmid. 
     
     
       18. The method of  claim 17 , wherein the DNA is transcribed to yield a ribonucleic acid. 
     
     
       19. The method of  claim 18 , wherein the ribonucleic acid is translated to yield a biologically active polypeptide. 
     
     
       20. The method of  claim 14 , wherein the polypeptide is at least one member selected from the group consisting of cyclosporin, Angiotensin I, II, or III, enkephalins and their analogs, ACTH, anti-inflammatory peptides I, II, or III, bradykinin, calcitonin, beta-endorphin, dinorphin, leucokinin, leutinizing hormone releasing hormone (LHRH), insulin, neurokinins, somatostatin, substance P, thyroid releasing hormone (TRH), and vasopressin. 
     
     
       21. The method of  claim 14 , wherein the antigen is at least one member selected from the group consisting of a membrane protein, a carbohydrate, envelope glycoproteins from viruses, an animal cell protein, a plant cell protein, a bacterial protein and a parasitic protein. 
     
     
       22. The method of  claim 14 , wherein the nutrient is at least one member selected from the group consisting of lycopene, vitamins, minerals, fatty acids, amino acids, fish oils, fish oil extracts, resveratrol, biotin, choline, inositol, ginko, saccharides, a phytochemical or zoochemical, beta-carotene, lutein, zeaxanthine, quercetin, silibinin, perillyl alcohol, genistein, sulfurophane, eicosapentanoic acid, gamma-3, omega-3, gamma 6 and omega-6 fatty acids. 
     
     
       23. The method of  claim 14 , wherein the vitamin is at least-one member selected from the group consisting of vitamins A, B, B1, B2, B3, B12, B6, B-complex, C, D, E, and K, vitamin precursors, caroteniods, and beta-carotene. 
     
     
       24. The method of  claim 14 , wherein the mineral is at least one member selected from the group consisting of boron, chromium, colloidal minerals, colloidal silver, copper, manganese, potassium, selenium, vanadium, vanadyl sulfate, calcium, magnesium, barium, iron and zinc. 
     
     
       25. The method of  claim 14 , wherein the saccharide or sweetener is at least one member selected from the group consisting of saccharine, isomalt, maltodextrine, aspartame, glucose, maltose, dextrose, fructose and sucrose. 
     
     
       26. The method of  claim 14 , wherein the flavor substance is an essential oil or an extract. 
     
     
       27. The method of  claim 26 , wherein the flavor substance is selected from the group consisting of oils and extracts of cinnamon, vanilla, almond, peppermint, spearmint, chamomile, geranium, ginger, grapefruit, hyssop, jasmine, lavender, lemon, lemongrass, marjoram, lime, nutmeg, orange, rosemary, sage, rose, thyme, anise, basil, black pepper and tea or tea extracts. 
     
     
       28. The method of  claim 26 , wherein the extract is from at least one member selected from the group consisting of an herb, a citrus, a spice and a seed. 
     
     
       29. The method of  claim 1 , further comprising introducing an aggregation inhibitor into the liposomal suspension. 
     
     
       30. The method of  claim 29 , wherein the aggregation inhibitor is at least one of casein, methylcellulose, albumin, serum albumin, bovine serum albumin, and rabbit serum albumin. 
     
     
       31. The method of  claim 1 , further comprising introducing an aggregation inhibitor to the cochleates. 
     
     
       32. The method of  claim 31 , wherein the aggregation inhibitor is at least one of casein, methylcellulose, albumin, serum albumin, bovine serum albumin, and rabbit serum albumin. 
     
     
       33. The method of  claim 13 , wherein the anti-fungal agent is Amphotericin B. 
     
     
       34. The method of  claim 2 , wherein the cargo moiety is added by dropwise addition. 
     
     
       35. The method of  claim 1 , wherein the cargo moiety is protonized by an acidic aqueous buffer or by slow addition of an acid. 
     
     
       36. The method of  claim 1 , wherein the negatively charged phospholipid is phosphatidylserine. 
     
     
       37. A method for administering a cochleate to a subject, the method comprising:
 administering the cochleate to the subject, wherein the cochleate is prepared by a process comprising the steps of: 
 (a) solubilizing a cargo moiety in a water miscible solvent; 
 (b) suspending a mixture of lipids in an aqueous solution to form a liposomal suspension comprising liposomes, wherein a majority of the mixture of lipids comprises a negatively charged phospholipid; 
 (c) combining the cargo moiety and the liposomes; 
 (d) precipitating the liposomes with a multivalent cation to form the cochleate comprising the cargo moiety; and 
 (e) removing the water-miscible solvent, 
  wherein the cargo moiety is protonized in any one or more of steps (a), (b), (c), and (d), by addition of an acid thereby lowering the pH of the solution below a maximum pK a  of the cargo moiety. 
 
     
     
       38. The method of  claim 14 , wherein the drug is Amphotericin B. 
     
     
       39. The method of  claim 15 , wherein the drug is amikacin.